1. Field of the Invention
The present invention, in the fields of virology and vaccine production, relates to replicated mammalian influenza viruses, grown in mammalian cell culture, which are suitable for use in mammalian influenza virus vaccine production. The replicated viruses are obtained from high growth strains of (i) reassortants between high growth master donor strains and clinical isolates, or (ii) passaged clinical isolates. The infected cell culture uses a low concentration range of trypsin (0.05-1.0 xcexcg/ml), continuously present in the medium, to provide high titers of the replicated virus. The invention also relates to methods for making and using such replicated viruses, such as for vaccine compositions and for vaccination methods.
2. Related Art
For the past several decades, fertilized chicken eggs have been used as a host system to replicate human influenza viruses with infectivity titers sufficient for use in vaccine production. Clinical isolates of human influenza virus are taken from infected patients and are reasserted in embryonated chicken eggs with laboratory-adapted master strains of high-growth donor viruses. The purpose of this reassortment is to increase the yield of candidate vaccine strains achieved by recombining at least the HA and NA genes from the primary clinical isolate isolates, with the internal genes of the master strain donor viruses. The high growth reassortant vaccine strains must also not contaminated with genes coding for antigenic determinants of the laboratory adapted viruses. This provides high growth reassortants having antigenic determinants similar to those of the clinical isolates. (Robertson et al., Biologicals 20:213-220 (1992)). The reassorted influenza virus is then grown in embryonated chicken eggs, purified from virus-containing allantoic fluid of the eggs and subsequently inactivated for use as vaccines.
However, a large body of data now suggests that this is a problematic system because of the frequency of viral mutation in antigenic sites of the major virus glycoprotein, hemagglutinin (HA), during replication in the chicken eggs. Even a single passage of a human influenza virus isolate or reassortant in chicken eggs leads to the selection of viral variants that differ in their antigenic determinants from those of the original clinical isolates. For example, the cultivation of influenza A and B viruses in chicken eggs often leads to the selection by the host system of variants having antigenic and structural changes in the viral HA molecule, making the variants ineffective or significantly less effective when used in an influenza vaccine (Kodihalli et al., J. Virol. 69:4888-4897 (1995); Gubareva et al., Virol. 199:89-97 (1994); Katz and Webster, J. Infect. Dis. 160:191-198 (1989); Wood et al., Virol. 171:214-221 (1989); Katz et al., Virology 156:386-395 (1987); Robertson et al., Virology 143:166-174 (1985)). In addition, the replicative properties of egg-grown viruses are not as consistent with natural infection as those of viruses grown in mammalian cells (Katz et al., J. Virol. 64:1808-1811 (1990); Robertson et al., Virology 179:35-40 (1990)).
Additionally, embryonated chicken eggs have potentially serious limitations as a host system, e.g., due to the lack of reliable year-around supplies of high-quality eggs and the low susceptibility of summer eggs to influenza virus infection (Monto, et al., J. Clin. Microb. 13:233-235 (1981)). Furthermore, the presence of adventitious agents in eggs can jeopardize the preparation of live-attenuated influenza virus vaccines. Adventitious agents are infectious contaminants (such as other viruses) in host systems that make them unsuitable or uncertifiable for use in vaccine production.
Cultured mammalian cells have also been used for virus replication and have been classified into at least two distinct groups. Primary diploid cells are those derived from intact tissue and have not been subcultivated. Continuous cell lines (CCLs) are cultured primary cells that replicate indefinitely and may be capable of growth in suspension culture. Haylick, in Continuous Cell Lines as Substrates for Biologicals, Arlington, Va., p. 2 (1988).
At present, many viral vaccines other than influenza are produced using primary trypsinized cells, including cells from monkey kidneys, and the kidneys of rabbits and hamsters. Primary diploid cell cultures have certain advantages such as easy preparation using simple media and bovine sera and sensitivity to a wide-range multiple viruses. However, primary diploid cells suffer from disadvantages, such as contamination by various adventitous agents, variable quality and sensitivity; and difficulty in obtaining suitable tissue for cultivation (e.g., monkey kidneys).
For example, primary diploid cell cultures obtained from monkey kidneys of wild animals usually contain endogenous viruses (Grachev, In Burgasov; ed., xe2x80x9cGuidance for the Production of Vaccines and Sera.xe2x80x9d Medicine, Moscow, p 176 (1978)). The number of adventitous agents depends on many factors, such as the methods of isolation, the cell systems used, the number of passages, the time of incubation and co-cultivation. The frequency of isolation of viruses from primary diploid cell cultures of monkey kidneys is directly proportional to the incubation period of the cells. Grachev, In Zh. Microbiol. Epidemiol. Immunobiol. 2:76 (1987).
In contrast, the advantages of using continuous cell lines are their retention of original antigenic characteristics of the infected virus, standardization, high susceptibility to variants of the same virus, and ability to be grown as a large mass of cells using microcarrier or suspension fermentor systems.
However, these advantages themselves do make such cell lines suitable for use in vaccine production. Mizrahi, ed., Viral Vaccines, Wiley-Liss, New York (1990), pp. 39-67. For example, influenza A viruses isolated and passaged exclusively in mammalian cell cultures have been found in some cases to retain most or all of their original antigenic characteristics, a feature that would prove highly advantageous in vaccine production. (Katz et al., Virology 165:446-456 (1988); Robertson et al., Virology 179:35-40 (1990); Katz et al., J. Infect. Dis. 160:191-198 (1989); Wood et al., Virology 171:214-221 (1989)).
However, mammalian primary diploid cell cultures present difficulties as a host system for vaccine production. This is due to problems such as contamination of the cell culture with adventitious agents, variable quality of the cells in the cell culture, different sensitivities of the cells to variants of the same virus, low virus titers and the high cost and difficulties in obtaining and preparing such cell cultures. In another example, although human diploid (MRC-5) cells can support the growth of influenza viruses, such systems have stringent growth media requirements making them suboptimal for large-scale production of influenza viruses for use in vaccines.
Furthermore, only MDCK cells, among the continuous cell lines tested, have been reported to support potentially sufficient growth and isolation of viruses (Frank et al., J. Clin. Microb. 10:32-36 (1979); Schepetink and Kok, J. Virol. Methods 42:241-250 (1993)). However, this line has been found to produce tumors and has thus not been certified for vaccine production, as not substantially free of adventitious agents.
Two other continuous cell linesxe2x80x94African green monkey kidney (Vero) cells and baby hamster kidney (BK-21)xe2x80x94are characterized, approved and certified by the World Health Organization (WHO) for production of human vaccines. However, Vero cells, while certified, were previously found unsuitable for large-scale production of human influenza virus vaccines. For example, the growth of influenza B in Vero cells was greatly restricted as compared to MDCK cells (Nakamura et al., J. Gen. Virol. 56:199-202 (1981)). Additionally, attempts to use Vero cells to evaluate the rimantadine sensitivity of human H1N1 and H3N2 influenza A viruses gave ambiguous results, due to the low titers of viruses produced in these cells, as compared with MDCK cells (Valette et al., Antimicrobiol. Agent and Chemotherapy 37:2239-2240 (1993)).
Thus, these and other studies indicate that influenza viruses have not previously replicated well in Vero cells, making them unsuitable for large-scale vaccine production. (Demidova et al., Vopr. Virosol (Russian) 346-352 (1979); Lau and Scholtissek, Virology 212:225-231 (1995)).
Itoh et al., Japan. J. Mol. Sci. Biol. 23:227-235 (1970) discloses a study of Vero cell cultures for infection with parainfluenza and influenza A viruses using a final added trypsin concentration of 1.5-1.8 xcexcg/ml. Itoh used chicken egg-grown viruses (Itoh et al. Virus 18:214-226 (1968)) to infect Vero cells under the above conditions, and found enhanced viral titers (over non-trypsin containing medium) for some of the influenza A strains tested, but Itoh could not demonstrate enhanced viral titers for influenza B strains.
Influenza viruses have eight negative-sense RNA (nsRNA) gene segments that encode at least 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (HA, active with cleavage and association of subunits HA1 and HA2), the matrix proteins (M1 and M2) and the non-structural proteins (NS1 and NS2) (Krug et al., xe2x80x9cExpression and Replication of the Influenza Virus Genome,xe2x80x9d in The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York (1989), pp. 89-152).
Influenza viruses are enclosed by lipid envelopes, derived from the plasma membrane of the host cell. The HA and NA proteins are the primary antigenic determinants and are embedded in the viral envelope by sequences of hydrophobic amino acids (Air et al., Structure, Function, and Genetics 6:341-356 (1989); Wharton et al., xe2x80x9cStructure, Function, and Antigenicity of the Hemagglutinin of Influenza Virusxe2x80x9d in The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York (1989), pp. 153-174).
The major influenza virus glycoprotein, HA, is synthesized in infected cells as a single polypeptide. Post-translational protease cleavage of the precursor HA results in the formation of the two subunits, HA1 and HA2, joined by a disulfide bond. Cleavage is essential for production of infectious viruses: virions containing uncleaved HA are noninfectious. The cleavage process can occur intracellularly or extracellularly. While HAs of infectious viruses are cleaved by extracellular proteases, such as from intestinal bacteria or the pancreas in vivo, the HAs of human, swine and most avian influenza virus strains cannot be cleaved by intracellular proteases. Therefore, replication of these viruses in many cell cultures requires the addition of a protease (such as trypsin) to the maintenance medium to ensure HA cleavage, thereby permitting activation of the progeny virus so that the rounds of infection can continue. Previous publications have suggested using a trypsin concentration in the range of 4-25 xcexcg/ml (U.S. Pat. No. 4,500,513). Vero cells were recently discovered to produce an activity that inactivates exogenous trypsin (Kaverin and Webster, J. Virol. 69:2700-2703 (1995)).
The background art has thus established a long-felt need for a method of influenza virus and vaccine production in a host cell system that improves on the use of chicken eggs. Such a host system would maintain antigenic properties of the clinical isolates of the natural virus and provide high titers of replicated virus, without having significant adventitious agents which are unsuitable for vaccine production.
The present invention includes methods, replicated viruses and vaccine compositions using high growth strains of mammalian influenza viruses, passaged from, or reasserted with, viral clinical isolates. The high growth strains of the invention can be reassortants made with laboratory high growth master donor strains, or they can be isolates that have been passaged in primary cell culture and selected for high growth. These methods use mammalian host cells that are infected with the high growth strains and then cultured with a continuous and low trypsin concentration in the culture medium. The methods provide replicated virus having high infectivity titers. These replicated, high growth strains are suitable for, and included in, influenza virus vaccines of the invention, for which the replicated virus is inactivated and/or attenuated.
The invention thus provides replicated influenza viruses and vaccines that comprise at least one replicated influenza virus strain, where the vaccine contains the replicated virus in an inactivated or attenuated form. These vaccines have substantially similar antigenicity to the viral clinical isolates, relative to chicken egg-grown viruses, where selection pressures in the eggs change the viruses"" antigenicity from that of the clinical isolates, such as through mutation of the HA gene.
In contrast to influenza viruses grown in other host cell types, such as chicken eggs, replicated influenza viruses and vaccines of the present invention provide at least one of: (i) substantially similar antigenicity to the clinical isolate; (ii) consistently high titers; (iii) lack of contamination by adventitious agents; (iv) consistent cell growth qualities; and (v) relatively less cost and technical difficulties in host cell replication.
Influenza virus vaccines of the invention can include at least one replicated virus strain (e.g., 1-50 strains) of a mammalian influenza virus A or B. Preferably the mammal host cells are continuous cell cultures of primary, cultured epithelial cells or fibroblasts, as mammalian cell lines of passage number 10-250. Preferably used for vaccine production are primary Vero cells as a continuous cell culture of a passage number of about 20-250. Currently available and certified (e.g., by the World Health Organization, WHO). Vero cell lines are passage number 135-190 (e.g., ATTC NO:X38).
Replicated influenza virus of the invention, in isolated, purified or concentrated form, preferably has an infectivity titer of about 106-109 (such as 106-107, 107 and 108-109, or any range or value therein) plaque forming units (PFU) per ml.
It is now discovered that providing a continuous, low trypsin concentration in the cell culture can circumvent the problem of trypsin inactivation or viral replication inhibition found previously in mammalian cell cultures. Such trypsin concentrations are also discovered to ensure multicycle replication that is comparable, in some or all respects, to that seen with either human influenza A or B viruses grown in chicken eggs.
The present invention also provides vaccine compositions comprising at least one strain of a replicated influenza virus of the present invention, in inactivated or attenuated form, optionally further comprising at least one of: (a) at least one pharmaceutically acceptable carrier or diluent; (b) at least one adjuvant and/or (c) at least one viral chemotherapeutic agent. The at least one carrier, diluent, adjuvant or chemotherapeutic agent enhances at least one immune response to at least one pathogenic influenza virus in a mammal administered the vaccine composition.
The present invention also provides a method for eliciting an immune response to at least one influenza virus strain in a mammal, which response is prophylactic or therapeutic for an influenza virus infection. The method comprises administering to the mammal a vaccine composition comprising an inactivated and/or attenuated, replicated influenza virus of the present invention. The composition is provided in an amount that is protective or therapeutic for the mammal against a clinical influenza virus pathology caused by infection with at least one influenza A or B virus strain.
Other objects, features, advantages, utilities and embodiments of the present invention will be apparent to skilled practitioners from the following detailed description and examples relating to the present invention.